Radiopharmaceuticals are drugs containing a radionuclide and are used routinely in nuclear medicine department for the diagnosis or therapy of various diseases. They are mostly small organic or inorganic compounds with definite composition. They can also be macromolecules, such as antibodies and antibody fragments that are not stoichiometrically-labeled with a radionuclide. Radiopharmaceuticals form the chemical basis for nuclear medicine, a group of techniques used for diagnosis and therapy of various diseases. The in vivo diagnostic information is obtained by intravenous injection of the radiopharmaceutical and determining its biodistribution using a gamma camera. The biodistribution of the radiopharmaceutical depends on the physical and chemical properties of the radiopharmaceutical and can be used to obtain information about the presence, progression, and the state of disease.
Radiopharmaceuticals can be divided into two primary classes: those whose biodistribution is determined exclusively by their chemical and physical properties; and those whose ultimate distribution is determined by their receptor binding or other biological interactions. The latter class is often called target-specific radiopharmaceuticals.
In general, a target specific radiopharmaceutical can be divided into four parts: a targeting molecule, a linker, a bifunctional chelator (BFC), and a radionuclide. The targeting molecule serves as a vehicle, which carries the radionuclide to the receptor site at the diseased tissue. The targeting molecules can be macromolecules, such as antibodies. They can also be small biomolecules (BM): peptides, peptidomimetics, and non-peptide receptor ligands. The choice of biomolecule depends upon the targeted disease or disease state. The radionuclide is the radiation source. The selection of radionuclide depends on the intended medical use (diagnostic or therapeutic) of the radiopharmaceutical. Between the targeting molecule and the radionuclide is the BFC, which binds strongly to the metal ion via several coordination bonds and is covalently attached to the targeting molecule either directly or through a linker. Selection of a BFC is largely determined by the nature and oxidation state of the metallic radionuclide. The linker can be a simple hydrocarbon chain or a long poly(ethylene glycol) (PEG), which is often used for modification of pharmacokinetics. Sometimes, a metabolizable linker is used to increase the blood clearance and to reduce the background activity, thereby improving the target-to-background ratio.
The use of metallic radionuclides offers many opportunities for designing new radiopharmaceuticals by modifying the coordination environment around the metal with a variety of chelators. The coordination chemistry of the metallic radionuclide will determine the geometry of the metal chelate and the solution stability of the radiopharmaceutical. Different metallic radionuclides have different coordination chemistries, and require BFCs with different donor atoms and chelator frameworks. For “metal essential” radiopharmaceuticals, the biodistribution is exclusively determined by the physical properties of the metal chelate. For target-specific radiopharmaceuticals, the “metal tag” may have significant impact on the target uptake and biodistribution of the radiopharmaceutical. This is especially true for metalloradiopharmaceuticals based on small molecules since in many cases the metal chelate contributes greatly to the overall size and molecular weight. Therefore, the design and selection of the BFC is very important for the development of a new diagnostic or therapeutic radiopharmaceutical.
Metallic radionuclides, such as 99mTc, 117mSn, 111In, 67Ga, 68Ga, 89Zr, and 64Cu, have been proposed for diagnostic imaging. Nearly 80% of radiopharmaceuticals used in nuclear medicine are 99mTc-labeled compounds. The reason for such a preeminent position of 99mTc in clinical use is its favorable physical and nuclear characteristics. The 6 hour half-life is long enough to allow those skilled in the art to carry out radiopharmaceutical synthesis and for nuclear medicine practitioners to collect useful images. At the same time, it is short enough to permit administration of millicurie amounts of 99mTc radioactivity without significant radiation dose to the patient. The monochromatic 140 KeV photons are readily collimated to give images of superior spatial resolution. Furthermore, 99mTc is readily available from commercial 99Mo—99mTc generators at low cost.
One of the characteristics of technetium is its rich and diverse redox chemistry. As of yet, there is no effective chemistry that can be used to attach the pertechnetate anion to a small biomolecule. Therefore, the Tc(VII) in 99mTcO4− has to be reduced to a lower oxidation state in order to produce a stable 99mTc-biomolecule complex or to a reactive intermediate complex from which 99mTc can be easily transferred to the BFC-BM conjugate.
A BFC can be divided into three parts: a binding unit, a conjugation group, and a spacer (if necessary). An ideal BFC is that which is able to form a stable 99mTc complex in high yield at very low concentration of the BFC-BM conjugate under mild conditions. There are several requirements for an ideal BFC. First, the binding unit can selectively stabilize an intermediate or lower oxidation state of Tc so that the 99mTc complex is not subject to redox reactions; oxidation state changes are often accompanied by transchelation of 99mTc from a 99mTc-BFC-BM complex to the native chelating ligands in biological systems. Secondly, the BFC forms a 99mTc complex that has thermodynamic stability and kinetic inertness with respect to dissociation. Thirdly, the BFC forms a 99mTc complex with a minimum number of isomers since different isomeric forms of the 99mTc-chelate may have significant impact on the biological characteristics of the 99mTc-BFC-BM complex. Finally, the conjugation group can be easily attached to the biomolecule.
For receptor-based radiopharmaceuticals, injection of large amount of BFC-BM may result in receptor site saturation, blocking the docking of the 99mTc-labeled BFC-BM, as well as unwanted side effects. In order to avoid these problems, the concentration of the BFC-BM in the radiopharmaceutical composition should be low. Otherwise, a post-labeling purification is often needed to remove excess unlabeled BFC-BM, which is time consuming and thus not amenable for clinical use. Therefore, the BFC attached to the biomolecule should have very high radiolabeling efficiency in order to achieve high specific activity.
For 99mTc-labeling of biomolecules, bifunctional chelators include N2S2 diaminedithiols, N2S2 diamidedithiols, N2S2 monoaminemonoamidedithiols, N3S monoaminediamidethiols, N3S triamidethiols, and 6-hydrazinonicotinamide (HYNIC). Various 99mTc-labeling techniques have been described in recent reviews (Liu et al., Bioconjugate Chem. 1997, 8, 621; Hom, R. K. and Katzenellenbogen, J. A. Nucl. Med. Biol. 1997, 24, 485; Dewanjee, M. K. Semin. Nucl. Med. 1990, 20, 5; Jurisson, et al., Chem. Rev. 1993, 93, 1137; Dilworth, J. R. and Parrott, S. J. Chem. Soc. Rev. 1998, 27, 43; Liu, et al., Bioconj. Chem. 1997, 8, 621; Liu, et al., Pure & Appl. Chem. 1991, 63, 427; Griffiths, et al., Bioconj. Chem. 1992, 3, 91; Liu, S. and Edwards, D. S. Chem. Rev. 1999, 99, 2235; Jurisson, S. and Lydon, J. D. Chem. Rev. 1999, 99, 2205). After radiolabeling, the resulting reaction mixture may optionally be purified using one or more chromatographic methods, such as Sep-Pack or high performance liquid chromatography (HPLC). The preferred radiolabeling procedures are those in which the chelation can be achieved without post-labeling purification.
Rhenium has two isotopes (186Re and 188Re) which might be useful in tumor therapy. 186Re has a half-life of 3.68 days with •-emission (Emax=1.07 MeV, 91% abundance) and a gamma-photon (E=137 keV, 9% abundance) which should allow imaging during therapy. 188Re has a half-life of 16.98 hours with an intense •-emission (Emax=2.12 MeV, 85% abundance) and 155 keV gamma photons (15% abundance). The related chemistry, medical applications, and radiolabeling with 186/188Re by direct and indirect methods have been reviewed (Fritzberg, et al., Pharmaceutical Res. 1988, 5, 325; Dilworth, J. R. and Parrott, S. J. Chem. Soc. Rev. 1998, 27, 43). Since the rhenium chemistry is very similar to technetium chemistry due to the periodic relationship, the methods used for the radiolabeling of biomolecules with 99mTc should apply to that with 186/188Re.
U.S. Pat. Nos. 5,206,370 and 5,753,520 disclose the use of organic hydrazines, such as HYNIC, and hydrazides as BFCs to modify proteins for labeling with radionuclides. For labeling with 99mTc, the hydrazino-modified protein is reacted with a reduced technetium species, formed by reacting [99mTc]pertechnetate with a reducing agent in the presence of a dioxygen chelating agent. The technetium is bonded through what are believed to be hydrazino or diazenido linkages. Since HYNIC moiety only occupies one or two coordination sites of the Tc, the coordination sphere of the Tc is often completed by the use of coligands, such as glucoheptonate and lactate.
U.S. Pat. No. 5,350,837 discloses a series of functionalized aminocarboxylates and their use as coligands in the radiolabeling of hydrazino-modified proteins. The improvements are manifested by shorter reaction times and higher specific activities for the radiolabeled protein. The best reported example is tricine.
U.S. Pat. No. 6,329,513 discloses a series of 99mTc complexes having a ternary ligand system containing a hydrazino or diazenido ligand, a phosphine ligand and a halide, in which the substituents on the hydrazido or diazenido ligand and phosphine ligand can be independently varied. However, the radiopharmaceuticals in this disclosure are formed in low specific activity. This disclosure shows no evidence that the halide ligand is indeed bonded to the metal center at the tracer (99mTc) level. The disclosure does not teach or suggest how to achieve the superior control of biological properties that will result from a ternary ligand system in which the substituents on the three types of ligands can be independently varied. Therefore, there remains a need for new ternary ligand systems, which form radiopharmaceuticals with high specific activity.
The advantage of using HYNIC as the BFC is its high labeling efficiency (rapid and high yield radiolabeling) and the choice of various coligands such as tricine and glucoheptonate, which allows easy modification of the hydrophilicity and pharmacokinetics of the 99mTc-labeled small peptides. For the 99mTc-labeling of HYNIC-conjugated small biomolecules, however, the use of tricine or glucoheptonate as coligands suffers two major drawbacks: the solution instability of binary technetium complexes [99mTc(HYNIC-BM)(L)2] (BM=biomolecule; L=tricine or glucoheptonate) in the absence of excess coligand, and the presence of multiple species of these complexes in solution due to different bonding modalities of HYNIC and the tricine or glucoheptonate coligands (Liu, et al., Bioconjugate Chem. 1996, 7, 63; Edwards, et al., Bioconjugate Chem. 1997, 8, 146; Edwards, et al., Bioconjugate Chem. 1999, 10, 884).
To increase the solution stability and minimize the number of isomeric forms in the [99mTc]HYNIC complexes, U.S. Pat. No. 5,744,120 discloses a ternary ligand system comprising an HYNIC-conjugated biomolecule (HYNIC-BM) as a ligand, tricine and a water-soluble phosphine as coligands. The new ternary ligand system forms ternary ligand 99mTc complexes in good yield and with high solution stability and a minimal number of isomeric forms. The Tc:HYNIC-BM:L:tricine (L=water-soluble phosphine) ratio was determined to be 1:1:1:1 through a series of mixed ligand experiments (Edwards, et al., Bioconjugate Chem. 1997, 8, 146), and has been confirmed by the FAB (fast-atom bombardment) and LC-MS data (Liu, et al., Inorg. Chem. 1999, 38, 1326; Liu, et al., Bioconjugate Chem. 2000, 11, 113).
U.S. Pat. No. 5,879,659 discloses novel ternary ligand 99mTc complex radiopharmaceuticals comprising an imine-N heterocycle, such as pyridine derivatives, as one of the three ligands. The imine-N containing heterocycles include, but are not limited to, nicotinic acid (NIC), isonicotinic acid (ISONIC), 4-pyridineacetic acid (PA), 4-pyridineethylsulfonic acid (PES), and 3-pyridinesulfonic acid (PSA). Like water-soluble phosphines, the imine-N containing heterocycles in this disclosure also form ternary ligand 99mTc complexes, [99mTc(HYNIC-BM)(tricine)(L)] (L=an imine-N containing heterocycle), as equal mixtures of two isomers. The Tc:HYNIC:L:tricine (L=imine-N containing heterocycle) ratio was determined to be 1:1:1:1 through a series of mixed ligand experiments, and has been confirmed by the LC-MS spectral data (Liu, et al., Bioconjugate Chem. 1998, 9, 583; Liu, et al., Bioconjugate Chem. 2000, 11, 113).
Like that of water-soluble phosphines, the bonding of the imine-N containing coligand to the Tc reduces the number of isomeric forms of [99mTc]HYNIC complexes, and increases the solution stability of 99mTc radiopharmaceuticals. Compared to water-soluble phosphines (TPPTS, TPPDS, and TPPMS), these imine-N heterocycles are much smaller so that the impact of the technetium chelate on receptor binding might be minimized. The hydrophilicity of [99mTc]HYNIC complexes can be tuned by changing the substituents on the heterocyclic ring. Neutral imine-N containing heterocyclic coligands are particularly interesting because they form neutral ternary ligand 99mTc complexes, which are able to cross the cell membranes and bind to intracellular receptors.
U.S. Pat. No. 6,251,364 discloses novel ternary ligand 99mTc complex radiopharmaceuticals comprising highly functionalized pyridine analogues, such as N-(2-hydroxyethyl)isonicotinamide (ISONIC-HE), 1-isonicotinamido-D-sorbitol (ISONIC-Sorb), and isonicotinamido-L-aspartic acid dimethyl ester (ISONIC-Asp-OMe2). The functionalized pyridine coligand is used to control the physicochemical and biodistribution characteristics of the ternary ligand 99mTc complexes. The extent of such control is dependent on the degree of functionalization of the pyridine coligand.
ISONIC-Asp-OMe2 is of particular interest because it contains two ester groups, which can be enzymatically hydrolyzed in biological systems. Since ISONIC-Asp-OMe2 carries no charge, it is expected to form neutral technetium complexes, which can cross the cell membrane and potentially bind to intracellular receptors, if the HYNIC-conjugated biomolecule carries no charge. Once inside the cell, enzymatic hydrolysis of these two ester groups will result in formation of negatively charged 99mTc-species, which cannot be easily washed out off the cell. In this way, the target uptake may be significantly improved. If ester groups are hydrolyzed in the blood stream, the negatively charged 99mTc-species is expected to have faster renal clearance than the neutral precursor. Therefore, the introduction of two enzymatically active ester groups offers two potential advantages: increasing target uptake and decreasing background.
The use of this ternary ligand system offers several advantages. The bonding of phosphine coligand to the Tc dramatically reduces the number of isomeric forms of the [99mTc]HYNIC complexes. The solution stability of [99mTc]HYNIC complexes is dramatically improved. The hydrophilicity of [99mTc]HYNIC complexes can be tuned either by altering the number of sulfonato groups or by using water soluble phosphines with other functionalities. The tricine coligand can also be substituted by other aminocarboxylates, such as dicine (N-bis(hydroxymethyl)methylglycine) and bicine (N,N-bis(hydroxymethyl)glycine). However, the specific activity of [99mTc]HYNIC complexes using bicine as coligands is not as high as that of the corresponding tricine complexes.
In principle, this ternary ligand system is useful for the 99mTc-labeling of any HYNIC-conjugated biomolecules. However, problems may arise when they are used for 99mTc-labeling of small biomolecules containing one or more disulfide linkages, which are often vital to keep the rigid cyclic conformation of the biomolecule and to maintain the high receptor binding affinity. The use of a large amount of water-soluble phosphine coligand, such as TPPTS, in combination with high temperature heating, which is needed for high-yield radiolabeling of hydrazone-protected HYNIC-conjugates, may destroy the S-S disulfide bonds and cause adverse effect on the biological properties of the 99mTc-labeled HYNIC conjugate.
Therefore, there is a continuing need for new ligand systems, which can be used for the 99mTc-labeling of small biomolecules (peptides or nonpeptide receptor ligands, enzyme inhibitors, or enzyme substrates) with high labeling efficiency, and that form 99mTc complexes with high stability and minimal isomerism. This invention is directed towards meeting this and other important needs.